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Seasonal Variations in Hydrogen Cyanide Concentration of Three Lotus Species

^a Dep. of Agron., Univ. of Missouri, Columbia, MO 65211
^b USDA-ARS, Plant Genet. Res. Unit, Columbia, MO 65211

Corresponding author (beuselinckp{at}missouri.edu)

Received for publication July 28, 2000.

	ABSTRACT

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Cyanogenic glucosides, generally considered antinutritional factors, are important defense molecules against predators and, in some cases, diseases. The objectives of this study were: (i) to determine the seasonal variations in hydrogen cyanide (HCN) concentration of three widely grown Lotus spp. and (ii) to assess the overall cyanogenic potential of the different plant components of a rhizomatous cultivar of broadleaf birdsfoot trefoil [Lotus corniculatus L.] (BFT). In this study, we used BFT cultivars Norcen and ARS-2620, narrowleaf trefoil (L. glaber Mill.) germplasm ARS-1207, and big trefoil (L. uliginosus Schkur.) germplasm ARS-1221. The experiments were conducted in the field and greenhouse using a randomized complete block design. Significant seasonal variations in HCN concentrations in Norcen, ARS-2620, and ARS-1207 were observed. Hydrogen cyanide concentrations were greatest in spring and summer and least in winter. ARS-1221 was acyanogenic. Of the three cyanogenic entries grown in the field study, ARS-1207 had the greatest concentration of HCN, averaging 900 µg g^-1 dry matter while Norcen and ARS-2620 had similar levels of HCN. In the greenhouse, Norcen and ARS-1207 had greater HCN concentrations than ARS-2620. Partitioning of the rhizomatous BFT cultivar ARS-2620 demonstrated that leaves and flowers produced the greatest concentration of HCN, five times as much as stems and ripe-seed pods. Rhizomes, which are typically produced in winter and fall, did not exhibit HCN production. Seeds of Norcen and ARS-2620 were acyanogenic, but ARS-1207 seeds were weakly cyanogenic. However, as seeds germinated and seedlings formed cotyledons, Norcen, ARS-2620, and ARS-1207 exhibited HCN. Roots of all species were acyanogenic.

Abbreviations: BFT, birdsfoot trefoil • DM, dry matter

	INTRODUCTION

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
FORAGE LEGUMES produce a wide range of metabolites implicated in defense mechanisms against predation, competition, and disease. Cyanogenic glycosides are defense molecules commonly found in Lotus spp. Cyanogenic glycosides release hydrogen cyanide (HCN) on either enzymatic or nonenzymatic hydrolysis (Compton and Jones, 1985). In Lotus spp., cyanogenesis is based on the cyanoglycosides linamarin and lotaustralin (Jones, 1977). Because the enzymes and their cyanogenic substrates are located in different compartments of the plant cell, no HCN is released from intact cyanogenic plants (Gruhnert et al., 1994). Thus, hydrolysis of the glycosides occurs after the tissues have been disrupted by herbivores, mechanical means, or fungal attack (Poulton, 1990). Acyanogenic (HCN negative) Lotus plants or plant parts may lack the cyanoglycosides, the corresponding ß-glucosidase, or both (Kakes, 1991; Compton and Jones, 1985).

The major role of cyanogenic glycosides in birdsfoot trefoil (BFT) is as a feeding inhibitor. In laboratory studies, several species of insects and mollusks preferred acyanogenic birdsfoot trefoil leaves or petals to the cyanogenic alternatives (Compton and Jones, 1985). As reviewed by Compton and Jones (1985), there is evidence that, in natural populations, acyanogenic plants suffer disproportionate levels of herbivory. Similar results have been reported for white clover (Trifolium repens L.) based on field and laboratory experiments (Hughes, 1991; Ellsbury et al., 1992). Furthermore, cyanogenesis has long been considered a resistance factor against fungi. Sorghum [Sorghum bicolor (L.) Moench] root exudates containing 0.6 to 2.7 mg kg^-1 CN inhibited chlamydospore germination of two Fusarium spp. (Hillocks et al., 1997). Yet, the presence of large amounts of cyanogenic compounds can inhibit active defense reactions in plants (Lieberei et al., 1989). Another possible use of cyanogenic glycosides in plants is as a storage form of reduced N (Poulton, 1990; Selmar et al., 1990), which may be important for winter survival and early spring growth.

Cyanogenesis is a genetically controlled character. Two independent genes are involved in the production of HCN: Ac/ac for the production of the cyanoglycosides and Li/li for the production of ß-glucosidase (Hughes, 1991). Cyanogenic Lotus plants have the phenotype AcLi (Bazin et al., 1994). Even though cyanogenesis is genetically controlled, its expression is often influenced by stress and other abiotic factors (Jones, 1988; Vickery et al., 1987; Pederson et al., 1996). In greenhouse experiments on white clover, Vickery et al. (1987) reported that HCN concentration was reduced by high light intensity, high temperature, and P application. Kakes (1991) suggested including representatives samples of populations and testing different tissues collected at various times in the growing season to develop a better understanding of cyanogenesis.

The perennial Lotus spp., broadleaf BFT, narrowleaf trefoil, and big trefoil, are used in major forage production centers of the world (Papadopulos and Kelman, 1999). Birdsfoot trefoil is the most important in North America, covering more than one million ha (Blumenthal and McGraw, 1999; Beuselinck and Grant, 1995). The three species markedly differ in morphology. Birdsfoot trefoil is a tetraploid (2n = 2x = 24) distributed throughout the world with a wide range of environmental adaptation. Big trefoil is a rhizomatous diploid (2n = 2x = 12) with a narrow geographic distribution and is particularly well adapted to low, wet, and waterlogged habitats (Papadopulos and Kelman, 1999). Narrowleaf trefoil is also a diploid (2n = 2x = 12) with a woody tap root and tolerates infertile, acidic, and poorly drained soils (Beuselinck and Grant, 1995).

A major problem limiting the use of BFT in the USA is stand persistence. Breeding and management efforts have been made to improve its persistence (Beuselinck et al., 1984; Li and Beuselinck, 1996). The development of a rhizomatous BFT cultivar should improve persistence by producing new plants vegetatively and replacing diseased or dead plants (Li and Beuselinck, 1996). Cyanogens as defense molecules have not been studied to determine their role in the persistence of BFT. It has been reported that, in other plant species, cyanogenesis could be a factor for fungal resistance (Hillocks et al., 1997) or susceptibility (Lieberei et al., 1989). Understanding the degree of cyanogenesis and its seasonal variation in Lotus spp. and cultivars may aid ongoing efforts to resolve problems of persistence.

The objectives of this research were: (i) to determine the level of HCN production and its seasonal variations in shoots and roots of the Lotus spp. BFT, narrowleaf trefoil, and big trefoil and (ii) to assess the overall cyanogenic potential of the different plant components of ARS-2620, a rhizomatous cultivar of BFT.

	MATERIALS AND METHODS

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Plant Establishment and Sample Processing
Mechanically scarified seeds of BFT cultivars Norcen and ARS-2620 (Beuselinck and Steiner, 1996), narrowleaf birdsfoot trefoil germplasm ARS-1207 (Steiner and Beuselinck, 2000b), and big trefoil germplasm ARS-1221 (Steiner and Beuselinck, 2000a) were planted in greenhouse pots containing a commercial peat-based media. Seedlings were inoculated with commercial Rhizobium strains (Urbana Lab., St. Joseph, MO) and allowed to grow under natural lighting for 12 wk. Seedlings were transplanted at the University of Missouri Agronomy Research Farm near Columbia, MO into a Mexico silt loam (fine, montmorillonitic, mesic Udollic Ochraqualf) soil the 2nd wk of May in 1996 and 1997. Plantings were made in a randomized complete block design with three replications with species as treatments. In each replication, there were 32 and 40 plants per treatment in 1996 and 1997, respectively. Plants were spaced 1 m between rows and 0.5 m within a row. Plants were sampled four times in 1996–1997—early fall (21 Oct.), late fall (11 Dec.), spring (10 June), and summer (25 Aug.)—and five times in 1997–1998—early fall (14 Oct.), late fall (12 Dec.), late winter (16 Mar.), spring (10 June), and summer (2 Sept.). Five plants from each cultivar and/or species were randomly chosen, dug, and washed free of soil, and shoots were mechanically separated from roots. Underground rhizomes were also collected from ARS-2620 in late fall and winter. All samples were packed individually in ice and transported to the laboratory. Samples were kept frozen at -4°C until freeze dried.

ARS-2620, a rhizomatous cultivar of BFT, was used to determine the concentration of HCN in different plant parts. Plants were randomly chosen from a field of ARS-2620 established in 1996 at the Agronomy Research Farm. Five plants were collected in mid-July 1998 and early August 1999. Each plant was separated into the components: leaves, floral parts (umbels), green seed pods, ripe brown seed pods, and stems. Herbage components from each plant were individually frozen at -4°C and later freeze-dried.

Greenhouse Study
In 1998 and 1999, seeds of Norcen, ARS-2620, ARS-1207, and ARS-1221 were planted in mid-April into 1-L plastic pots filled with commercial growth medium as previously described. Each entry was replicated eight times. Seedlings were thinned to one plant per pot and allowed to grow for 4 mo under natural lighting in the greenhouse. The pots were watered as needed. Herbage was clipped to a height of 5 cm in mid-August, frozen at -4°C, and freeze-dried.

All freeze-dried plant samples from the field and greenhouse were ground with a Udy cyclone mill (Model 3010-018, Udy Corp., Fort Collins, CO) to pass a 1-mm screen. Ground samples were stored in sealed containers at -20°C until analyzed for HCN.

Seed and Seedling Investigation
Approximately 3 g of Norcen, ARS-2620, ARS-1207, and ARS-1221 seeds were crushed to a fine powder in a mortar and pestle with dry ice. The crushed seeds were tested in four replications for the presence or absence of HCN by the modified picrate paper method (Vickery et al., 1987). Seeds of the four entries were also germinated in mid-June in the greenhouse under natural lighting to determine the onset of cyanogenesis. Seedlings with developed cotyledons were harvested 3 d after germination. Ten fresh seedlings of each entry per replication were dipped in liquid N₂ and crushed in a test tube with a glass rod. Crushed seedlings were assayed for the release of HCN in four replications.

Laboratory Analysis
Ground tissue and seed samples (200 mg) were placed in 250-mL flasks sealed with a rubber stopper. Five mL of water and 2 to 3 drops of chloroform (CHCl₃) were added, and the flask was swirled to break up clumps and distribute the sample evenly in liquid. The release of HCN was measured as described by Vickery et al. (1987). Samples were incubated at room temperature for 48 h. The enzymatic release of HCN was indicated by a color change of picrate paper from yellow to reddish brown. Test strips were removed from flasks and placed in clean test tubes, and the color was eluted in 10 mL double-distilled water and compared with a series of standards using Potassium cyanide (KCN) and sodium picrate solution. Color intensity was read with a Spectronic Genesys 5 spectrophotometer (Spectronic, Rochester, NY) at 625-nm wavelength. The color intensity of the test strips was also visually scored using a scale of 0 to 5 where 0 indicated no color change (acyanogenic), 1 was light red (weakly cyanogenic), and 5 was deep red-brownish color (strongly cyanogenic). For samples that did not exhibit a positive HCN reaction, approximately 1 mg of the enzyme ß-glucosidase g^-1 of tissue was added to ensure a complete release of HCN if it was present in the tissue (Vickery et al., 1987).

Statistical Analyses
Data were analyzed using the general linear model of SAS version 6.0.3 (SAS Inst., 1988). The 1996–1997 and 1997–1998 HCN field data were separately analyzed because number of harvests in each set of the study were unequal. The greenhouse data for the 2 yr were combined. Similarly, the 2 yr of field data of ARS-2620 plant components were combined. In both cases, there were no significant year x treatment interaction, and the error variances were homogenous. Means were separated using Fisher's protected LSD and considered to be significant at P 0.05.

	RESULTS AND DISCUSSION

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Field Study
Shoot samples of ARS-2620, Norcen, and ARS-1207 were highly cyanogenic in both years and all seasons, with release of HCN evident within 15 min after the picrate test was initiated. ARS-1221 was completely acyanogenic. The results demonstrated HCN concentration of the cyanogenic Lotus entries varied greatly among species and seasonal samplings (Fig. 1 and 2), and there was a significant species x season interaction both years. Roots of all species were acyanogenic. Addition of ß-glucosidase and extension of the incubation period of the root samples to 72 h did not produce detectable HCN. Regardless of the concentration of HCN in the aboveground plant parts, roots of these species were consistently acyanogenic.

View larger version (37K): [in this window] [in a new window]   Fig. 1. Seasonal variation in HCN concentration of field-grown Lotus spp. Values are means of Norcen, ARS-2620, and ARS-1207. Bars having the same letter are not significantly different at P = 0.05

View larger version (46K): [in this window] [in a new window]   Fig. 2. Seasonal variation in HCN concentration of Norcen, ARS-2620, and ARS-1207 grown in the field. Vertical bars represent SE among species within a season

Among the four entries studied, ARS-1207 consistently had the greatest concentration of HCN in the fall, spring, and summer seasons, averaging 900 µg g^-1 (Fig. 2). However, samples taken in winter of 1998 demonstrated that the HCN concentrations in ARS-1207 and BFT were similar. During winter sampling, the plants were in initial stages of breaking dormancy and had not yet developed many leaves. This reduced physiological state could have resulted in low HCN production and minimized differences among entries. Norcen and ARS-2620 generally had similar levels of HCN, with a slight increase noted during the summer season for ARS-2620. Jones (1977) proposed that an individual BFT plant could be acyanogenic at some times of the year and cyanogenic at others. This phenotypic instability was due to variable amounts of cyanoglycoside and enzyme in response to changes in temperature (Ellis et al., 1977). In our study, the three cyanogenic entries, ARS-2620, Norcen, and ARS-1207, remained cyanogenic during all samplings while ARS-1221 remained acyanogenic. ARS-1221 is high in condensed tannins, which are negatively correlated with HCN (Bazin et al., 1994; Gebrehiwot and Beuselinck, 1997). Tannins are known to inhibit enzyme activity (Hagerman and Butler, 1981; Goldstein and Spencer, 1985). So high concentration of tannins in ARS-1221 could have reduced HCN expression. In papaya (Carica papaya L.), it has been shown that tannin precipitates several enzymes, including added ß-glucosidase (Goldstein and Spencer, 1985). Thus, care is needed in interpreting negative HCN tests in light of possible ecological synergisms between plant defense chemicals.

Variation in HCN production between and within a population of Lotus spp. is not an uncommon feature. Grant and Sidhu (1967) reported the association between HCN content and basic chromosome number. They found a greater concentration of HCN in Lotus spp. with a basic chromosome number of n = 7 than in the species with n = 6. In our study, even though all entries had a basic chromosome number of n = 6, ARS-1207 was strongly cyanogenic and ARS-1221 completely acyanogenic. ARS-1221 herbage samples were analyzed for HCN with added ß-glucosidase and were found to be negative for HCN, indicating that ARS-1221 lacked the required glucoside. The diploid narrowleaf trefoil is a putative progenitor of the tetraploid BFT (Steiner, 1999). The strongly cyanogenic character of ARS-1207 and the HCN level of Norcen and ARS-2620 support that hypothesis.

Greenhouse Study
The greenhouse study confirmed the cyanogenic nature of ARS-2620, Norcen, and ARS-1207 vs. the acyanogenic ARS-1221. In contrast to the field study, Norcen had a greater concentration of HCN than ARS-2620 and was similar to ARS-1207 (Fig. 3). Among other factors, the high concentration of HCN in Norcen could probably be due to greater proportion of leaf tissue and flowers compared with the other entries. Norcen is later flowering than ARS-2620 and ARS-1207 and maintained more leaves and flowers under greenhouse conditions in July and August. Patterns of cyanogenic polymorphism are not similar throughout the genus Lotus, and there are no common responses to a given set of environmental conditions (Band et al., 1981). Furthermore, environmental conditions can suppress or effect cyanogenesis in plants that have cyanogenic potential (Aikman et al., 1996). Thus, it is not surprising that the HCN concentration of field and greenhouse grown plants differs in magnitude or ranking.

View larger version (33K): [in this window] [in a new window]   Fig. 3. Hydrogen cyanide concentration of Norcen, ARS-2620, and ARS-1207 grown in the greenhouse for 16 wk. Values are means of 2 yr. Bars having the same letter are not significantly at P = 0.05

View larger version (26K): [in this window] [in a new window]   Fig. 4. Concentration of HCN in different plant components of ARS-2620 birdsfoot trefoil (BFT) grown in the field and harvested in midsummer. Values are means of 2 yr. Bars having the same letter are not significantly different at P = 0.05

It generally appears that the HCN is placed strategically according to the plants need for defense, i.e., greater concentration where the plant component is most vulnerable to predators. Briggs and Schultz (1990) reported the change in defense chemical concentrations in BFT as plants mature and progress into floral and seed development. They found that overall HCN concentration was depressed when plants produced fruits.

Cyanogenesis of Seeds and Seedlings
Seeds of ARS-2620, Norcen, and ARS-1221 were acyanogenic with or without the addition of ß-glucosidase to the picrate test (Table 1). Seeds of ARS-1207 were weakly cyanogenic. It is not uncommon for Lotus seeds to be HCN negative. Band et al. (1981) reported that ripe pods and seeds of 12 Lotus spp., including BFT, were acyanogenic. When seeds were germinated and the cotyledons developed, HCN was detected. Three-day-old seedlings of ARS-2620, Norcen, and ARS-1207 were positive for HCN production (Table 1). These findings agree with Grant and Sidhu (1967), who reported that cotyledons of various Lotus seedlings reacted positively to the picrate test when seedlings were exposed to light. Seedlings of ARS-1221 remained acyanogenic (score = 0) even with the addition of ß-glucosidase to the picrate test.

The presence or absence of HCN in roots and rhizomes may have significant importance in disease resistance and plant persistence. Sorghum root exudates containing HCN have been known to inhibit the germination of chlamymdospores of Fusarium oxysporum in the laboratory and in the rhizosphere (Hillocks et al., 1997). Fungal pathogens, primarily Fusarium spp., are responsible for severe stand reduction in BFT (Gotlieb and Doriski, 1983; English, 1999). Identifying Lotus genotypes with cyanogenic root systems as a means to improve persistence merits further investigation. However, it should be noted that the BFT cultivars Norcen and ARS-2620 are susceptible to Rhizoctonia solani foliar blights during the hot and humid conditions of late-summer at the field research site where our study was conducted. It appears that cyanogenesis in these cultivars of BFT is not sufficient to render substantial protection against Rhizoctonia solani.

In conclusion, the sodium picrate test is sensitive enough to group Lotus spp. based on the degree of cyanogenesis. Because the expression of cyanogenesis is affected by environmental factors, plant age, growth phase of the plant, and the plant part used for the test (Aikman et al., 1996), it is important to pay attention to these factors when making comparisons. In this study, Lotus seed and root samples were not reliable for characterizing cyanogenesis. Lotus leaves or shoots are more reliable for assessing cyanogenesis. Although Norcen, ARS-2620, and ARS-1207 expressed a high degree of cyanogenesis, no animal toxicity has been reported in these Lotus spp. Generally, plants that accumulate >600 µg HCN g^-1 DM pose potential danger to livestock (Haskins et al., 1987). However, because most animals have the ability to detoxify the cyanide in forages upon ingestion (Wheeler, 1994), toxicity occurs only under exceptional circumstances. Despite a high level of HCN accumulation in their shoots, Norcen, ARS-2620, and ARS-1207 lack detectable levels of HCN in their roots. Identifying or developing BFT with roots positive for HCN merits consideration in tackling root disease susceptibility of BFT.

	NOTES

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Contrib. of the Missouri Agric. Exp. Stn. Journal Ser. no. 13056. Mention of a trademark, vendor, or proprietary product does not constitute a guarantee or warranty of the product by the USDA or the Univ. of Missouri and does not imply its approval to the exclusion of other products or vendors that may also be suitable.

	REFERENCES

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